PROJECT SUMMARY Poly(ADP-ribose) polymerase 1 (PARP1) is a promising target for cancer therapy in DNA repair-compromised tumors. One PARP1 inhibitor (Olaparib) is used in the clinic to treat ovarian cancer, and ~20 phase III clinical trials with inhibitors of PARP1 are currently ongoing. PARP1 is a highly abundant nuclear protein that upon binding damaged DNA adds long chains of poly(ADP-ribose) onto itself and many other nuclear proteins. This baroque post-translational modification recruits key components of the DNA repair machinery. Existing inhibitors of PARP1 require a high effective dose (100-400 mg/day) and show poor correlation of drug efficacy with inhibition of PARP1 activity, possibly because they also target inactive PARP1 that is abundantly bound to undamaged chromatin. This PARP1 population has many nuclear functions that extend beyond the maintenance of genome integrity, such as chromatin compaction, and the regulation of transcription and DNA methylation. Our goal is to use rigorous quantitative, structural, and mechanistic approaches to investigate how PARP1 interacts differently with damaged vs. intact DNA in the context of chromatin. We aim to understand if selective inhibition of the DNA-bound active conformation of PARP1 is a potential approach for increasing drug efficacy and specificity. Using novel enzymatic assays, we will test the varied outcomes of activation by different types of DNA damage. We also propose to investigate the interplay between PARP1 and the much less active and less abundant PARP2. Our studies will reveal the conformational diversity of PARP1 and PARP2, and show how this affects the complex reaction mechanism, resulting in different downstream signaling events. Our mechanistic studies will provide key insights into how to design better screens and assays for the development of more selective, mechanism-based PARP inhibitors. Altogether our proposed research will aid the development of the next generation of PARP inhibitors for use in cancer therapy.